week 1 quiz

Which of the following is used to standardize the bacterial inoculum used in a disk diffusion test (Kirby-Bauer method)?

A 0.5 McFarland Standard is used to prepare a sample for a standard disc diffusion (Kirby-Bauer) susceptibility test. The 0.5 McFarland standard is also used for the preparation of inocula in standardized agar dilution, broth macro and microdilution, and anaerobic organism susceptibility test procedures.

The susceptibility of a bacterium to each drug on a sensitivity plate using antibiotic disks is demonstrated by the diameter of the zone of:

A zone of inhibition is the area around an antibiotic-infused paper disk that does not show any bacterial growth. The antibiotic impregnated on the disk will diffuse into the agar in the area surrounding the disk. If the bacteria are sensitive to the antibiotic, they cannot grow near the disk. The size of the zone is proportional to how sensitive the organism is. If the organism is resistant to the antibiotic, it will grow very closely to the disk.

Which of the following antibiotics inhibits protein synthesis by binding to the 30S ribosomal subunits and provides coverage against Gram positive and Gram negative aerobes?

Aminoglycosides

A beta-hemolytic Streptococcus was isolated from a throat culture. It gave the following biochemical reactions: Bacitracin - Susceptible Bile esculin - Negative 6.5% NaCl - No growth CAMP - Negative SXT - Resistant

Beta hemolytic Streptococcus, Group A

Which of the following enzymes, produced by Staphylococcus aureus is responsible for preventing penicillin antibiotics from being effective?

Beta-lactamase is the enzyme, produced by S. aureus, that is responsible for preventing penicillin antibiotics from being effective. Beta-lactamase breaks down the beta-lactam antibiotics, e.g., penicillins, cephalosporins, carbapenems and monobactams allowing S. aureus to be resistant to these antibiotics. Catalase distinguishes staphylococci from streptococci. Staphylococci are catalase positive and streptococci are catalase negative. The production of coagulase distinguishes S. aureus from other staphylococci. S.aureus is coagulase positive, while other staphylococci are coagulase negative. Hyaluronidase aids S. aureus in skin colonization and infection spread.

A patient grew out a Staphylococcus lugdunensis and after susceptibility testing, the isolate gave results indicating probable resistance to oxacillin. Which of the following is the best method to detect the mec-A gene to determine if the organism has true oxacillin resistance?

Cefoxitin disk diffusion is the correct answer because the use of a cefoxitin disk can detect the presence of the mecA gene in Staphylococcus aureus, Staphylococcus lugdunensis, and coagulase negative staphylococcus. For Staphylococcus aureus and Staphylococcus lugdunensis, if zone growth is = 21mm, this indicates mecA positive. If zone growth is = 22mm, the organism is mecA negative. For coagulase negative staphylococci, zone growth = 24 mm indicates mecA positive. Zone growth = 25 mm, the organism is mecA negative.

The upper image is a photomicrograph of a Gram stain prepared from a positive blood culture obtained from a 68 year old female with an indwelling urinary bladder catheter. Smooth, gray, faintly beta hemolytic colonies were recovered in subcultures of the positive bottle. The tubes shown in the lower photograph are: an esculin slant (left) displaying intense brown coloration a 6.5% sodium chloride broth (right) showing cloudy growth

Enterococcus faecalis is the correct answer. The Gram stain illustrates gram-positive cocci in pairs and short chains consistent with Enterococcus species. The hydrolysis of esculin and ability to grow in 6.5% sodium chloride are characteristics consistent with that species. Enterococcus faecalis can be alpha, beta, or gamma hemolytic on sheep blood agar.

Fluoroquinolones, such as ciprofloxacin, work by targeting:

Fluoroquinolones target topoisomerases, enzymes that control DNA replication processes. Penicillin and other ß-lactam antibiotics are cell wall synthesis inhibitors which target penicillin-binding proteins. Sulfonamides are DNA synthesis inhibitors which disrupt the folic acid pathway. Protein synthesis inhibitors, such as macrolides, work by binding to 50S ribosomes.

An alpha-hemolytic streptococcus isolated from a urine culture is resistant to optochin and bactracin, grows in 6.5% NaCI broth, and is PYR positive. The organism is MOST likely:

Growth in 6.5% NaCl broth and PYR positive indicates the most likely identification is an Enterococcus species. This organism may be described as alpha-hemolytic but has also been described as gamma-hemolytic and occasionally beta-hemolytic. The organism may be optochin negative but this test is not typically performed on alpha-hemolytic organisms from a urine sample. Bacitracin is usually performed on beta-hemolytic organisms. Streptococcus pyogenes is PYR positive but it is strongly beta-hemolytic, bacitracin sensitive, does not grow in high salt concentration. It is not typically associated with urinary tract infections. Streptococcus agalactiae may be isolated from urine cultures but it is beta-hemolytic and occasionally gamma-hemolytic. It is bacitracin resistant, PYR negative, and will not grow in high salt concentration. Streptococcus pneumoniae is not associated with urinary tract infections. This organism is alpha hemolytic but it is optochin sensitive, PYR negative, and it does not grow in high salt concentrations.

All of the following organisms have capsular material as a defense mechanism

Haemophilus influenzae Streptococcus pneumoniae Neisseria meningitidis

A microbiology laboratory recently purchased a rapid identification instrument that uses a laser on the microorganisms, which produces an ion cloud. These ions are then separated in a tube and travel within the tube to a detector. The time of travel will be measured and a mass spectrum will be produced. Which of the following techniques uses this methodology?

MALDI-TOF is the correct answer because the name describes its methodology (Matrix-Assisted Laser Desorption Ionization Time of Flight mass spectrometry). With this methodology, an organism will be placed directly onto a plate and a chemical matrix will be applied. Afterwards, a laser will be applied to the sample and the matrix will absorb the energy creating an ion cloud. These ions will travel through a tube called a flight tube. The lighter ions will travel faster through the tube to the detector while the heavier ions will travel slower. The detector will measure the time of flight and report out a mass spectrum. This spectrum will be compared to other spectrums within the data library for identification.

Micrococcus luteus

Micrococcus luteus colonies are smooth, but often produce distinctly yellow pigment. Tetrad formation of the gram-positive cocci may be observed microscopically in gram stains. The catalase reaction is positive, and the coagulation reaction is negative. Confirmation of M. luteus can be made with further testing, such as positive modified oxidase, and susceptibility to bacitracin ("A disc").

Which of the following is a key benefit that molecular methods can offer over culture methods?

Molecular diagnostic tests provide for the rapid detection of certain microorganisms that are difficult to culture or take a long time to grow on culture media, thereby reducing the turnaround time in case of a life-threatening situation. In most cases, molecular methods do represent an increased cost per test, due to the cost of reagents and instrument platforms. Also, molecular methods may provide better sensitivity than traditional culture methods.

Some strains of Group F Strep can carry the group A antigen, potentially leading to a false positive identification of group A, Streptococcus pyogenes. A spot test that is helpful in differentiating these two species and thus preventing a false report is:

Most strains of group A Streptococcus pyogenes are PYR positive, and therefore can be differentiated from other beta hemolytic streptococci, all of which are negative. All of the streptococci are catalase negative; therefore, this is not a helpful discriminating test. Bile solubility is a test that is helpful in distinguishing Streptococcus pneumoniae (bile soluble) from all other streptococci, which are bile resistant. The streptococci do not possess cytochrome oxidase activity; therefore, this is not a discriminatory test.

Complete hemolysis of sheep blood agar as demonstrated by the image below would be seen in which of the following catalase-negative isolates?

Strep pyogenes produces colonies that are about 0.5 mm in diameter, appear translucent with a smooth surface and have a beta-hemolytic zone that is up to four times the diameter of the colony. Streptococcus pneumoniae is alpha-hemolytic which demonstrate a green area of partial hemolysis around the colonies. Staphylococcus aureus will produce beta-hemolysis, but is catalase positive. Enterococcus faecium generally produces alpha-hemolytic colonies, but can also produce non-hemolytic colonies.

Rothia mucilaginosa

Rothia mucilaginosa colonies are clear to light pink-gray and are distinctly mucoid and adherent to the agar surface. Rothia spp. may resemble gram positive coryneform bacilli, in pairs or short, filamentous chains, but gram positive cocci are observed when grown in broth. A definitive identification requires further testing. Rothia is also indigenous to the oral cavity and may be recovered in cases of dental plaque infections.

Which of the following organisms is most likely pictured in the Gram stain photomicrograph taken from a broth?

Staphylococcus aureus appears as gram positive cocci in clusters.

Staphylococcus aureus

Staphylococcus aureus colonies are smooth and may be cream-colored or yellow, but can be microscopically differentiated from both S. epidermidis and Micrococcus sp. by observing gram-positive cocci that form tight grape-like clusters rather than arranging in loose clusters or in tetrads. Confirmation can be made by demonstrating a positive coagulase reaction

Examine the following test results and identify what action you would take. CAMP: Arrowhead appearance Bacitracin disk: 6mm zone of inhibition PYR: Negative Bile Esculin: Tan appearance, no darkening

Streptococcus agalactiae is the most likely organism because of the positive CAMP test (an arrowhead of beta hemolysis is positive; no arrowhead is negative).

The organism in the attached photos was isolated from a sputum specimen. The blood agar plates were incubated at 37° C. The upper photo shows 24 hour incubation and the lower photo shows 48 hour incubation. The Gram stain showed lancet-shaped Gram positive cocci in pairs. What is the most likely identification of this organism?

Streptococcus pneumoniae is the correct response. The large mucoid appearing colonies indicate the synthesis of capsular polysaccharide characteristic of S. pneumoniae. Narrow zones of alpha hemolysis may be observed. This identification is further supported by observing the colony auto-hydrolysis after prolonged incubation, resulting in sunken centers in what have been referred to as "checker pieces". The presumptive identification is supported by the characteristic lancet-shaped Gram positive cocci in pairs on the Gram stain.

The VDRL and RPR are used to provide presumptive identification of which infection?

Syphilis Treponema pallidum

Beta-lactam antibiotics interfere with cell wall synthesis by all of the following, EXCEPT:

Targeting the 30S ribosomal subunit Transpeptidase enzymes catalyze the final stage of peptidoglycan synthesis during cell wall formation. Beta-lactam antibiotics bind with these enzymes, interfering with their function. The transpeptidase enzymes are also referred to as PBP's, or penicillin-binding proteins. Aminoglycosides and tetracyclines interfere with the 30S ribosomal subunit (protein synthesis inhibitors).

Abiotrophia defectiva (formerly Streptococcus defectivus) and Granulicatella adiacens (formerly S. adjacens, then A. adiacens) are the names currently given to two species of what were previously called nutritionally variant streptococci (NVS). What supplement must be added to blood culture medium to recover these organisms in cases of "culture negative endocarditis?"

The nutritionally deficient streptococci were also termed thiol-requiring or pyridoxal-requiring variants because they require thiol compounds, either cysteine or the active form of vitamin B6 (pyridoxal), for growth.

A D-test is performed on gram positive cocci to determine inducible resistance to clindamycin when the isolate is sensitive to clindamycin and resistant to which antibiotic?

When erythromycin tests resistant and clindamycin is susceptible, an isolate should be tested for inducible clindamycin resistance. A positive test is indicated by a blunting of the zone of inhibition between the two disks, demonstrating a "D" around the clindamycin disk, giving it the name, "the D-test". The D-test is based on a modified double disk diffusion test and is only used to detect inducible macrolide resistance, none of the other antibiotics listed are macrolides.

All of the following would be suitable for identification of bacterial antigens

agglutination reactions, immunofluorescence methods, and precipitin reactions

Polymerase chain reaction (PCR) copies DNA through repeated cycles of three basic steps. What is the correct order of these steps?

denaturation, annealing, extension. Extraction of the DNA from the clinical sample occurs before the initiation of PCR. Once extracted, the sample is heated to a temperature at which the strands of DNA separate (denaturation). Next, the temperature is lowered and the primers attach (anneal) to their complementary targets. Lastly, the temperature is raised and new strands of DNA are synthesized (extension), utilizing free nucleotides in the reaction mixture.

Oxacillin resistance in clinical strains of staphylococci is confirmed by the detection of _______ gene.

he gold standard for identification of oxacillin resistance in staphylococci is the detection of the mecA gene using commercial latex or molecular detection methods.

A methicillin-resistant Staphylococcus aureus was recovered from a wound infection. Which antibiotic would be used for treatment?

vancomycin

A mucoid alpha hemolytic translucent colony suspected of being Streptococcus pneumoniae was isolated from a blood culture. All of the of the following are appropriate tests to make a presumptive or definitive identification of this organism

Bile solubility test Specific co-agglutination assay Optochin susceptibiity Bacitracin susceptibility testing is used for the presumptive identification of Streptococcus pyogenes, not for Streptococcus pneumoniae. The bile solubility test can be used in the presumptive identification of S. pneumoniae. It is positive while other alpha-hemolytic Streptococcus spp. are negative. This test detects the lysis of S. pneumoniae in the presence of bile salts. A Phadebact pneumococcus test is available for the identification of S. pneumoniae. It is a co-agglutination assay. Streptococcus pneumoniae is susceptible to optochin while other alpha hemolytic Streptococcus spp. are not.

The condition most likely to predispose a person to septicemia by viridans streptococci is:

The oropharynx is thought to be the source in most cases of viridans streptococcus with organisms entering the tissue during dental or surgical procedures. Poor oral hygiene and periodontal disease may be the precursor to bacteremia with viridians strep especially in immunocompromised patients

MIC

minimum inhibitory concentration MIC is defined as the lowest concentration that inhibits visible growth. Methods for measuring the MIC include disk diffusion procedures (Kirby-Bauer), micro-broth dilutions and gradient diffusion (E-test strip). The MIC is used to determine the susceptibility or resistance of a specific isolate against specific antibiotics. All other options are not the intended definition for MIC.

A nasopharyngeal specimen is processed and fixed onto a microscope slide. Next, fluorescein-conjugated antibody is added to the slide. The specimen is incubated with the labeled antibody, washed, and then observed for fluorescence. Which of the following techniques best describes this process?

direct immunofluorescence or direct fluorescent antibody test (DFA) tests can be used to detect antigens of Legionella, Bordetella, Neisseria, and Treponema in appropriate clinical specimens. The specimen is fixed to a microscopic slide and fluorescein-conjugated antibody is added to the slide and incubated. If the corresponding antigens are present in the specimen, they will bind to the fluorescent antibody. DFA can detect only antigens, not antibodies. The method described is a DFA technique.

Illustrated in the photograph is a bile esculin agar plate inoculated with a bacterial isolate from a urinary tract infection. The most likely identification is:

A bacterial isolate recovered from the urinary tract that hydrolyzes esculin and grows in the presence of bile is most likely to be an Enterococcus species. Bacteroides fragilis also has the capability of growing in the presence of bile and hydrolyzing esculin; however, this species is an anaerobe that would not grow on the aerobic bile esculin agar used for the recovery of bacteria from urine specimens. Stenotrophomonas maltophilia hydrolyzes esculin; however, it does not grow in the presence of bile and therefore would not appear on bile esculin medium. Streptococcus mutans, one of the viridans streptococci, does not grow on bile esculin agar and does not hydrolyze esculin.

A patient is being treated with gentamicin for a Gram negative bacterial infection. The chemistry department has repeating peak and trough orders on the patient. Which of the following antibiotic classes does gentamicin belong?

Aminoglycosides is the correct answer because this group of antibiotics inhibits bacterial protein synthesis by irreversibly binding to protein receptors on the 30S ribosomal subunit. Blood levels for this group of antibiotics must be monitored to prevent nephrotoxicity. Examples of antibiotics in this class are gentamicin, tobramycin, amikacin, streptomycin, and kanamycin.

Enterococcus spp. are commonly resistant to all of the following antimicrobial drug types

Beta-lactams, Glycopeptides, Aminoglycosides* Chloramphenicol (from the phenicol drug type) is one of the agents given to patients with vancomycin resistant Enterococcus (VRE). Enterococcus spp. have acquired and intrinsic resistance to aminoglycosides, beta-lactams, and glycopeptides

A tech receives a call from the floor regarding antimicrobial therapy. The patient culture yielded an aerobic Gram-negative bacillus and the physician would like to use an antibiotic that inhibits DNA synthesis. Which of the following antibiotics would be the best choice?

Ciprofloxacin part of fluroquinoles.* Cefazolin is part of the Cephalosporin family and part of the beta-lactam antibiotics with the Penicillins. Cephalosporins have a species range of both aerobic and anaerobic gram-positive and gram-negative species. The main target for these antibiotics are penicillin binding proteins, which inhibits cell wall synthesis.

Staphylococcus epidermidis

Colonies are smooth with a white or gray-white color. Microscopic observation of gram stains reveals gram-positive cocci in small loose clusters, often in tetrad formation. The catalase reaction is positive, and the coagulation reaction is negative. A definitive identification requires further testing.

Penicillin G, ampicillin, carbenicillin, and methicillin are antimicrobial agents whose MAIN biological function is to:

Penicillin G, ampicillin, carbenicillin, and methicillin are ß-lactam antimicrobial agents whose MAIN biological function is to inhibit cell wall synthesis. ß-lactam antibiotics inhibit cell wall synthesis by binding to penicillin-binding proteins (PBPs) and destroy or inhibit peptidoglycan-based cell walls in both Gram-positive and negative species. Protein synthesis inhibitors target the 30S or 50S ribosomes, resulting in a variety of errors in protein synthesis. Examples of protein synthesis inhibitors include aminoglycosides, tetracyclines, and macrolides. Lipopeptides such as daptomycin or polymixin B disrupt the cell membrane in both Gram-positive and Gram-negative bacteria, preventing cell wall synthesis. DNA synthesis inhibitors include fluoroquinolones and sulfonamides, and provide activity against Gram-positive and Gram-negative species.

Observed on the surface of blood agar are smooth, white, 48-hour-old colonies incubated at 37oC, demonstrating a positive catalase reaction and a negative coagulase reaction. The sample was taken from an intravenous catheter. In the photomicrograph are gram positive cocci distinctly arranged in loose clusters and in tetrad formation. From these observations, select the presumptive identification of this isolate.

Staphylococcus epidermidis

This is a catalase-positive, coagulase-negative, gram-positive coccus isolated from a urine specimen from a 20-year-old female college student. The image shown is a Mueller Hinton plate streaked with a 0.5 MacFarland standardized inoculum and a 5 microgram disk of novobiocin after overnight incubation. What is the identification of the isolate?

Staphylococcus saprophyticus is the correct answer because this organism is resistant to novobiocin as evidenced by the lack of zone of inhibition around the novobiocin disk. It is a common cause of urinary tract infections in young sexually active females.

A presumptive identification of a bacterial species can often be made by observing the type of hemolysis when grown on 5% sheep blood agar. Which of the following organisms is nearly always alpha hemolytic?

Streptococcus pneumoniae is typically alpha hemolytic on 5% sheep blood agar. Streptococcus pyogenes is beta hemolytic. Enterococcus spp. can be alpha, beta, or gamma hemolytic, but is typically gamma hemolytic. Streptococcus agalactiae is beta hemolytic.

The small, transparent, gray-yellow 48 hour colonies incubated at 37°C. as illustrated in the upper photograph, are surrounded by wide zones of beta hemolysis. This culture was obtained from a throat swab of a patient with pharyngitis. The lower photograph demonstrates susceptibility of this isolate to the bacitracin "A" disc. What is the presumptive identification of this isolate?

Streptococcus pyogenes (Group A) will have the initial observation of relatively small transparent colonies surrounded by broad zones of beta hemolysis provides for a presumptive identification of Streptococcus Group A. This identification can be confirmed by demonstrating susceptibility to bacitracin, as observed by the wide clear zone surrounding the A disc.

Which of the following media is commonly used when performing the Kirby-Bauer disk diffusion?

The disc diffusion method for antibiotic susceptibility testing is the Kirby-Bauer method. For this procedure, the Mueller-Hinton preparation is the standard agar-base medium used for testing most bacterial organisms, although certain supplements and substitutions are required for testing fastidious organisms. Chocolate agar is commonly used for growing Neisseria gonorrhoeae and Haemophilus spp., so it can be used for antibiotic susceptibility testing for those organisms. Thayer-Martin agar is selective for N. gonorrhoeae and N. meningitidis, so it can be used for antibiotic susceptibility testing for those organisms. MacConkey agar is the most frequently used primary selective and differential agar, so it would not be used for antibiotic susceptibility testing. It will inhibit the growth of gram positive bacteria and fungi while allowing the growth of many types of gram negative bacilli. It can also demonstrate lactose fermentation of the bacteria by showing pink/red growth of the organisms. Non-lactose fermenters will remain colorless and translucent.

In this image is a quadrant plate containing brain heart infusion agar supplemented with 6 µg/mL of vancomycin. The right upper quadrant was inoculated with the test strain of Enterococcus faecium. The presence of growth in the inoculated quadrant indicates ___________?

The organism is resistant to vancomycin The presence of growth on the vancomycin screening plate indicates that this isolate is resistant to vancomycin, at least up to a level of 6 µg/mL, which is above the 4 µg/mL set by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) as the cut-off. In certain circumstances, it may be necessary to perform minimum inhibitory concentration (MIC) susceptibility studies to determine the phenotype of a resistant isolate.

Oxacillin resistance of MRSA is due to production of which of the following penicillin binding proteins (PBPs)?

The oxacillin resistance of MRSA is determined by the production of a novel penicillin-binding protein called PBP2a (PBP 2'), that does not bind oxacillin, rendering the drug ineffective. The mecA gene encodes the production of PBP2a. Latex agglutination kits are available to detect these altered PBPs, and could serve as an alternative method for testing oxacillin resistance.

This Gram stain was prepared from a lower respiratory tract specimen. How should this Gram stain be reported?

The smear is most accurately reported as "Many gram-positive cocci in pairs, many PMNs." It is important to quantitate and describe the morphology (bacilli, cocci, etc.) of the microorganisms that are observed as thoroughly as possible. Include the arrangement of the cells (pairs, clusters, etc.) and report the presence and quantity of host cells (PMNs, epithelial cells, etc.) that are observed on the smear.

The following is true of the disk diffusion method of measuring antimicrobial sensitivity:

provides an interpretation value of sensitive, intermediate, or resistant. The zone of inhibition is correlated to the broth or agar dilution MIC values. The disk diffusion method of antimicrobial susceptibility testing does not provide a semi-quantitative MIC value. The zone of inhibition surrounding the disk is measured in millimeters and correlated to the broth or agar dilution MIC value giving interpretations of sensitive, intermediate or resistant. The disk diffusion method requires standardization of pH, agar depth, inoculum concentration, disk concentration, cation concentration, and more. The disk diffusion method of antimicrobial testing is most frequently manually interpreted. Automation has been developed but it is not as commonly used as the automated MIC methods.

The photograph on the right is of a 5% sheep blood agar plate inoculated with a throat culture. It is common practice to stab the agar with the inoculating needle, as seen at the tip of the arrow, in order to detect or accentuate the reaction of:

stabbing the agar in areas of inoculation has the effect of submerging the growing bacteria under the surface of the agar, where conditions are relatively anaerobic. Oxygen labile hemolysin, or streptolysin O (SLO) is observed only under anaerobic conditions;

What are the two general categories of nucleic acid amplification (NAA) techniques?

target amplification and signal amplification. Target amplification and signal amplification are two general categories of amplification techniques. Target amplification involves making copies of a target sequence to such a level that they can be detected in vitro. Signal amplification does not increase the number of target or probe sequences, but does increase the amount of signal bound to target sequences. Polymerase chain reaction (PCR), RT-PCR, and strand displacement amplification (SDA) are specific examples of target amplification, while branched chain DNA (bDNA) is an example of signal amplification. FISH assays do not involve amplification.